By convention, the adenovirus life cycle is divided into two phases, the early and the late phase, which are separated by the onset of viral DNA replication and characterized by the expression of specific subsets of the viral genome. In the early phase, the early genes E1, E2, E3, and E4 are maximally expressed (E1b, E2, E3, and E4 expression being activated by the immediate early gene E1a (see, e.g., Berk et al., Annu. Rev. Genet., 20:45-79 (1986)), and the major late transcription unit (MLTU) is expressed at very low levels and is attenuated. At the onset of viral DNA replication, a marked switch in gene expression occurs, resulting in the reduction of expression of the early genes and an increase in activation of the late genes, most of which are translated from mRNAs originating from the MLTU.
The MLTU is 25 kB in length and contains six polyadenylation (polyA) sites, five that are unique to the MLTU (L1 through L5) and one used by both the E3 transcription unit and the MLTU (see, e.g., Prescott et al., Mol. and Cell. Biol., 17(4):2207-2216 (1997)). Each polyA site is associated with several potential splice acceptors, and all 5′ terminal exons are spliced to a common tripartite leader (see, e.g., Nevins et al., Adv. Virus Res., 26:1-35 (1981)). Due to complex alternative RNA splicing, each late region produces multiple mRNAs. Indeed, during the late stage of adenovirus infection, all six polyA sites are used in conjunction with a variety of splice acceptors to produce a minimum of 45 different mature mRNAs. The extremely high level of mRNA production and the novel splicing/polyadenylation capacity of the MLTU are essential to the production of adenoviral virions.
Recombinant adenoviral vectors characterized as having a whole or partial deletion of one or more early region genes have been extensively studied. Many of these recombinant adenoviral vectors are engineered to contain a nucleic acid sequence encoding a therapeutic factor prior to being delivered to a population of cells for gene therapy. Limitations remain, however, for the production of an adenoviral vector able to carry out its effects (e.g., to deliver a therapeutic factor) only in certain cell types (e.g., cancer cells). Progress has been made with these limitations in mind by employing a number of different approaches. Some of these approaches have investigated inserting non-native ligands, or nucleic acid sequences encoding such ligands, into the MLTU, which theoretically allows for these adenoviral vectors to bind specific cell surface receptors to which it may not normally bind. For example, International (PCT) Patent Application WO 99/55365 describes an adenoviral vector comprising a first non-native nucleic acid sequence and a second non-native nucleic acid sequence which is different from the first non-native nucleic acid sequence. The first non-native nucleic acid sequences encodes, for example, a chimeric fiber protein that does not bind to the native adenoviral fiber receptor known as the Coxsackievirus-Adenovirus Receptor (CAR) but will bind to a receptor present on the surface of a target cell of interest. The fiber protein which binds to a receptor present on the surface of a target cell can be placed at any suitable location within the adenoviral genome, including 3′ of the L5 polyA site. This fiber protein also comprises splice acceptor elements and 3′ polyA signals.
Similarly, U.S. Pat. No. 5,543,328 describes an adenovirus comprising a fiber protein comprising a ligand, which is specific for a receptor located on a desired cell type. The ligand either replaces a portion of the fiber protein, or the adenovirus includes a fusion protein composed of the adenovirus fiber protein and the ligand. In certain embodiments, the ligand can be a tumor necrosis factor (TNF), transferrin, ApoB, α-2-macroglobulin, α-1 acid glycoprotein, mannose-containing peptide, sialyl-Lewis-X antigen-containing peptide, CD34 ligand, CD40 ligand, ICAM-1, M-CSF, circumsporozoite protein, VLA-4, LFA-1, NGF, HIV gp120, Class II MHC antigen, colony stimulating factor (CSF), insulin-like growth factor, or Interleukins 1 through 14. The adenovirus also can include a gene(s) encoding a therapeutic factor(s), which is typically inserted into the E1 or E3 region of the adenovirus.
In other approaches, a nucleic acid sequence encoding a therapeutic factor or ligand is not included in the adenoviral genome at all. For example, U.S. Pat. No. 5,677,178 (McCormick-Onyx Pharmaceuticals) describes the use of a certain type of replication-deficient adenovirus that replicates in certain abnormal (i.e., cancer) cells but does not replicate in normal (i.e., non-cancerous) cells. The conditionally replication-deficient adenovirus kills the abnormal cells through the expression of a replicative phenotype wherein the abnormal cells are lysed as part of the viral replication process. Thus, the adenovirus is replication-deficient in normal cells but is replication competent in abnormal cells.
To achieve this selective replication, the adenoviral vector described in the '178 patent substantially lacks an expressed viral oncoprotein capable of binding a functional p53 tumor suppressor gene product and/or a functional Rb tumor suppressor gene product. Abnormal cells lacking a functional p53 tumor suppressor gene product and/or a functional Rb tumor suppressor gene product can support the replication of the otherwise replication-deficient adenovirus substantially lacking an expressed viral oncoprotein capable of binding a functional p53 tumor suppressor gene product and/or functional Rb tumor suppressor gene product, respectively.
While previously-described vectors have been somewhat effective at delivering nucleic acids to target cells or treating certain disease states, a need remains to provide an adenoviral vector which more selectively carries out its activities in target cells, i.e., cells responsible for certain disease states, such that these adenoviral vectors can be formulated into therapeutic compositions and used in methods of treating these disease states. The invention provides such a vector, composition, and method. These and other advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.